DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF AMLODIPINE BESYLATE AND IRBESARTAN

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1 Indexed in Cite Factor - Directory of International Research Journals in association with leading Universities DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF AMLODIPINE BESYLATE AND IRBESARTAN MAZHARUDDIN M. SHAIKH *1, STAVAN MASTER 2 & ABRAR M. CHAUDHARY 3 ABSTRACT The present work describes a simple, rapid, and reproducible reverse phase high performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Amlodipine besylate (AML) And Irbesartan (IRB). The separation was achieved on a Hypersil BDS C18 (250mm X 4.6 mm i.d., 5 μm particle size) with a gradient system of Water (ph3.5): ACN in the ratio of 60:40 v/v. The mobile phase at a flow rate of 1.0 ml/min, Injection volume 20μl and wavelength of detection used was 245 nm. The retention time for Amlodipine besylate and Irbesartan was obtained as ±0.1min and 5.133±0.2min, respectively. The linearity of the proposed method was investigated in the range of 5-15 μg/ml and μg/ml for Amlodipine besylate and Irbesartan, respectively. Correlation coefficient was and for Amlodipine besylate and Irbesartan. The developed method was validated as per ICH guideline, for its accuracy, precision, LOD & LOQ and the results were found to be satisfactory, thus the method is specific, rapid and simple with good sensitivity for estimation of Amlodipine besylate and Irbesartan. These analytical methods are also applicable in ordinary laboratories. It can also be adopted for quality control tests for these drugs in tablets. KEYWORDS Amlodipine besylate, Irbesartan, RP-HPLC method, Validation. *Address for correspondence AUTHORS AFFILIATION Department of Quality Assurance Techniques, Parul Institute of Pharmacy and research, Vadodara, India , mazharshaikh114@yahoo.com. 1

2 INTRODUCTION Amlodipine besylate is a long-acting 1,4-dihydropyridine calcium channel blocker. It acts primarily on vascular smooth muscle cells by stabilizing voltage-gated L-type calcium channels in their inactive conformation. By inhibiting the influx of calcium in smooth muscle cells, amlodipine prevents calcium-dependent myocyte contraction and vasoconstriction. A second proposed mechanism for the drug s vasodilatory effects involves ph-dependent inhibition of calcium influx via inhibition of smooth muscle carbonic anhydrase. Some studies have shown that amlodipine also exerts inhibitory effects on voltage-gated N-type calcium channels. N-type calcium channels located in the central nervous system may be involved in nociceptive signaling and pain sensation. Amlodipine is used to treat hypertension and chronic stable angina. Figure 1: Structure of Amlodipine besylate. Irbesartan 2-butyl-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]-1,3-diazaspiro[4.4]non- 1-en-4-one, Irbesartan is an angiotensin receptor blocker (ARB) used mainly for the treatment of hypertension. Figure 2: Structure of Irbesartan. Association of Amlodipine besylate with irbesartan can be especially interesting, due to the antihypertensive. It is used in patients with hypertension uncontrolled by usual doses of irbesartan or amlodipine besylate alone. The present study was designed to develop a simple, precise, and rapid analytical RP-HPLC procedure, which can be used for the analysis of assay method for simultaneous estimation of Amlodipine besylate and Irbesartan as there was only individual methods reported for both drugs. The combination of these two drugs is not official in any pharmacopoeia; hence no official method is available for the simultaneous estimation of these two drugs in their combined dosage forms. Literature survey of Amlodipine besylate and Irbesartan revealed several methods for detecting these drugs individually but there is no method for their simultaneous estimation using RP-HPLC. 2

3 The developed method was validated as per ICH guidelines and its updated international convention. The linearity of response, precision, ruggedness and robustness of the described method has been checked. Chemicals and Reagents EXPERIMENTAL Amlodipine besylate API was gifted by Osaka Pharmaceutical ltd, Vadodara, India. Irbesartan API was gifted by Cadila pharmaceutical limited, Ankleshwar, India. The combination of these drugs is manufactured by Dainippon Sumitomo Pharma Co Ltd. Japan. All the reagents including potassium dihydrogen phosphate and ortho phosphoric acid were purchased from Merck and Rankem Ltd. New Delhi, India. HPLC Instrumentation and Conditions The analysis was carried out on a HPLC system (Shimadzu-LC 20AT) equipped with UV detector, pressure controlled by prominence pump and operated by Spinchrom Software. C18 column (250 mm 4.6 mm i.d., particle size 5 μm) was used for separation. Mobile phase used for separation was containing Water: Acetonitrile in the ratio of 60:40 v/v adjusted to ph 3.5 with orthophosphoric acid. The flow rate was kept at 1.0 ml/min, column temperature was ambient (25 C), eluents were detected by UV detector at 245 nm, and the injection volume was 20 μl. Preparation of mixed standard stock solutions of Amlodipine besylate and Irbesartan 10 mg of standard Amlodipine besylate and 100 mg of standard Irbesartan were accurately weighed and transferred to a 100ml Volumetric flask and dissolved in 50ml of Diluent (Mobile phase). The flask was shaken and volume was made up to the mark with mobile phase to give solution containing 100 μg/ml of Amlodipine besylate and 1000 μg/ml of Irbesartan. Selection of detection wavelength In the present study individual drug solutions of 10μg/ml were prepared in different solvent mixtures of HPLC grade organic and inorganic solvents at different ratio. These drug solutions were then scanned in the UV region of nm and the spectrums were recorded to get λmax of analyte in Mobile Phase. 245nm wavelength was selected for estimation of this combination. Method Validation Linearity and Range The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in sample within a given range. The range of analytical method is the interval between the upper and lower levels of analyte that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity. The linearity peak area response was determined by analyzing solutions having concentrations in the range of 5-15 μg/ml and μg/ml for Amlodipine besylate and Irbesartan respectively from same solution. Peak area of each solution was measured using 3

4 developed method. Calibration curve of peak area Vs concentration was plotted. The correlation coefficient and regression line equations for Amlodipine besylate and Irbesartan were determined. Accuracy To study the accuracy powdered synthetic mixture analysis of the same was carried out. Recovery studies were carried out by addition of standard drug to the sample at 3 different concentration levels (80, 100 and 120 %) taking into consideration percentage purity of added bulk drug samples. It was determined by calculating the recovery of Amlodipine besylate and Irbesartan by standard addition method. Accuracy is the closeness of the test results obtained by the method to the true value. Preparation of sample solution for % recovery Marketed formulation equivalent to 10mg Amlodipine besylate and 100mg Irbesartan were accurately weighed and transferred to volumetric flask of 100 ml capacity and make up the volume up to 100 ml. From that transferred 1ml of an aliquot in to 10 ml volumetric flask to make final concentration 10 μg/ml Amlodipine besylate and 100 μg/ml Irbesartan and make the volume up to 10 ml. The resulting solution was filtered through Whatman filter paper. Absorbance of sample solutions was measured at selected wavelength of Amlodipine besylate and Irbesartan and concentration is calculated which is known as pre-analyzed sample. In pre-analyzed sample 80, 100 and 120 % of Amlodipine besylate and Irbesartan was spiked. Absorbance of spiked samples was measured and total amount of drug was calculated and from which % recovery was calculated. Precision Repeatability 6 replicates of standard mixture solution having Amlodipine besylate (10 μg/ml) and Irbesartan (100 μg/ml) were prepared and chromatograms were recorded and RSD were calculated. Intraday precision Standard solutions containing 5.0, 10.0 and 15.0 μg/ml Amlodipine besylate and 50, 100 and 150 μg/ml Irbesartan were analyzed 3 times on the same day as per the procedure. Chromatogram of each sample was taken. SD and RSD were calculated. Interday precision Standard solutions containing 5.0, 10.0 and 15.0 μg/ml Amlodipine besylate and 50, 100 and 150 μg/ml Irbesartan were analyzed on three different days as per the procedure. Chromatogram of each sample was taken. SD and RSD were calculated. Limit of Detection Calibration curve was repeated for 6 times and the standard deviation (SD) of the intercepts was calculated than LOD was calculated as follow from the formula: LOD= (3.3 SD)/Slope. 4

5 Where, SD = the standard deviation of Y- intercept of 6 calibration curves. Slope = the mean slope of the 6 calibration curves. Limit of Quantitation Calibration curve was repeated for 6 times and the standard deviation (SD) of the intercepts was calculated than LOQ was calculated as follow from the formula: Robustness Study LOQ= (10 SD)/Slope To evaluate robustness of the method few parameters were deliberately varied. The parameters included were variation of flow rate, change in ph of mobile phase and change of mobile phase ratio. The change was made at 3 levels and replicate for 3 times. The system suitability parameters were calculated for Amlodipine besylate and Irbesartan. Change in Flow Rate One concentration of mixture Amlodipine besylate(5μg/ml) and Irbesartan (66.5μg/ml) was analyzed 3 times at 3 different flow rate and record the peak area, resolution and tailing factor were recorded and percentage was R.S.D. calculated. Acceptance criteria: % RSD should be less than 2. Change in Mobile Phase Ratio One concentration of mixture Amlodipine besylate (5μg/ml) and Irbesartan (66.5μg/ml) was analyzed 3 times at 3 different mobile phase ratio and record the peak area, resolution and tailing factor were recorded and percentage R.S.D. was calculated Acceptance criteria: % RSD should be less than 2. Change in ph One concentration of mixture Amlodipine besylate (5μg/ml) and Irbesartan (66.5μg/ml) was analyzed 3 times at 3 different ph and record the peak area, resolution and tailing factor were recorded and percentage was R.S.D. calculated. Acceptance criteria: % RSD should be less than 2. System suitability 5 μg/ml Amlodipine besylate and 66.5 μg/ml Irbesartan in mixture was injected and the chromatograms was recorded. The resolution, number of theoretical plates, and peak asymmetry were calculated to determine whether the result complies with the recommended limit. Analysis of Pharmaceutical Preparations: %Assay of Amlodipine besylate and Irbesartan in their Tablet Dosage Form (Formulation: AIMIX Tablet) 5

6 Sample preparation For estimation of Amlodipine besylate and Irbesartan, 5 Tablets were taken and crushed, Powder accurately weighed. Powder equivalent to 10 mg Amlodipine besylate and 100 mg Irbesartan was accurately weighed and transferred to volumetric flask of 100ml capacity. 100 ml of methanol was transferred to this volumetric flask and sonicated for 15 min. The flask was shaken and volume was made up to the mark with methanol. The above solution was filtered through whatman filter paper (0.45μ). From this solution 10 ml was transferred to volumetric flask of 100 ml capacity. Volume was made up to the mark to give a solution containing 10 μg/ml Amlodipine besylate and 100 μg/ml Irbesartan. This solution was used for the estimation of Amlodipine besylate and Irbesartan RESULT AND DISCUSSION Optimization of Chromatographic Conditions To develop suitable RP-HPLC method for simultaneous estimation of Amlodipine besylate and Irbesartan, different chromatographic conditions were applied and optimized chromatographic conditions were developed.(see figure 3) Figure 3: Optimized chromatogram of Amlodipine besylate and Irbesartan. Optimized chromatographic conditions are as follows(see table 1) Table 1. Optimized chromatographic conditions: Parameters Stationary phase Condition Hypersil BDS C18 column (250mm X 4.6 mm i.d., 5 μm particle size) Mobile phase Water(pH-3.5): ACN (60 : 40) Pump mode Flow rate (ml/min) 1.0 Isocratic Run time (min) 10.0 Volume of injection (μl) 20 Detection wavelength (nm) 245 6

7 Validation Linearity Linearity was performed on synthetic mixture of Amlodipine besylate and Irbesartan as per marketed formulation composition.(see figure 4) Figure 4: Chromatograms of Amlodipine besylate and Irbesartan for Linearity. Calibration curve for the Amlodipine besylate (5-15 μg/ml) Chromatogram for following concentrations 5, 7.5, 10, 12.5 and 15 μg/ml for Amlodipine besylate at 245 nm at flow rate of 1 ml/min was obtained. Peak area were obtained as tabulated in Table 2 and the graph of calibration curve was obtained as shown in Figure 5. Table 2: Calibration curve for Amlodipine besylate. Conc (n=6) Area(mean ± S.D.) a r e a amlo y = 145.4x R² = Conc.(ppm) Figure 5: Graph of Calibration curve for Amlodipine besylate. 7

8 Discussion: Linearity range for Amlodipine besylate was found to be 5-15 µg/ml in Mobile Phase. Regression Equation for Amlodipine besylateat 245 nm: Y=145.4x r 2 value: Calibration curve for the Irbesartan ( μg/ml) Chromatogram for concentrations 50, 75, 100, 125 and 150 μg/ml for Irbesartan at 245 nm at flow rate of 1 ml/min. Peak area were obtained as tabulated in Table 3 and the graph of calibration curve was obtained as shown in Figure 6. Table 3: Calibration curve for Irbesartan Conc (n=6) Area(mean ± S.D.) a r e a irbe y = 35.68x R² = Conc.(ppm) Figure 5: Graph of Calibration curve for Irbesartan. Discussion: Linearity range for Irbesartan was found to be µg/ml in mobile phase. Regression Equation for Irbesartan at 245 nm.y= 35.68x , r 2 value: Accuracy (% Recovery study) Amlodipine besylate Concentration of preanalyzed sample of Amlodipine besylate: 8

9 Table 4: %Recovery data for Amlodipine besylate. %Spiking Amount of test taken Amount of std added Total amount of Conc. Total conc. found Calculated spiking Conc. Mean % Recovery ± SD 80 (n=3) (n=3) (n=3) ± ± ± (n=3 Determination) Irbesartan Concentration of preanalyzed sample of Irbesartan: Table 4. %Recovery data for Irbesartan %Spiking Amount of test taken Amount of std added Total amount of Conc. Total conc. Found Calculated spiking Conc. Mean % Recovery ± SD 80 (n=3) (n=3) (n=3) ± ± ± (n=3 Determination) 9

10 Precision Repeatability Table 5: Repeatability data for Amlodipine besylate and irbesartan. Drug Target Conc. Peak Area of Sample Mean SD %RSD Amlodipine besylate Irbesartan Discussion The % RSD for Repeatability of both the drugs was found to be less than 2. So, it was concluded that proposed method for estimation of Amlodipine besylate and Irbesartan is precised in nature. 10

11 Intraday precision Table 6: Intraday precision data for Amlodipine besylate and irbesartan. Drug Target Conc. Peak Area of Sample Mean SD %RSD Amlodipine besylate Irbesartan Discussion % RSD for Intraday precision of both the drugs was found to less than 2. So, it was concluded that proposed method for estimation of Amlodipine besylate and Irbesartan is precised in nature. 11

12 Interday precision Table 7 : Interday precision data for Amlodipine besylate and Irbesartan Drug Target Conc. Peak Area of Sample Mean SD %RSD Amlodipine besylate Irbesartan Discussion The % RSD for Interday precision of both the drugs were found to be less than 2. So, it was concluded that proposed method for estimation of Amlodipine besylate and Irbesartan is precised in nature. 12

13 Limit of Detection (LOD) and Limit of Quantitation (LOQ) Table 8: LOD and LOQ data for Amlodipine besylate and Irbesartan. Parameters Amlodipine besylate Irbesartan Mean Slope (n=6) SD of Y intercept (n=6) Discussion LOD LOQ The proposed method can quantify small amount of drugs with precisely. So, it was concluded that the proposed method is very sensitive in nature. Robustness Study Change in flow Rate Table 9: Robustness data for Amlodipine besylate and Irbesartan with change in flow rate. Parameter Amlodipine besylate(%rsd) Irbesartan(%RSD) Folw 0.2)0.8ml/min Flow 0.2)1.2ml.min rate(+ rate( Mobile phase(62:38) Mobile phase(58:42) ph(- 0.2) ph(+0.2) System suitability Table 10: System suitability Parameters Name Rt (min) Area Tf Resolution Theoretical Plate# Amlodipine besylate Irbesartan

14 Analysis of Pharmaceutical Preparations: %Assay of Amlodipine besylate (10μg/mL) and Irbesartan (100μg/mL) in their Tablet Dosage Form Figure 6: Chromatograph of 10 μg/ml of Amlodipine besylate and 100μg/mL of Irbesartan prepared from Tablet (AIMIX TAB) AIMIX TAB Label claim mg/tablet Conc. taken for assay Ave. Peak area of sample* Conc. Found form Tablet * % Assay* ± SD AMLO 10 mg IRBE 100 mg Discussion % Assay of Amlodipine besylate and Irbesartan was found in an acceptance limit (98-102%) so this method can be used for analysis of Amlodipine besylate and irbesartan in combined dosage form. CONCLUSION The proposed RP-HPLC method was used for the simultaneous estimation of Amlodipine besylate and Irbesartan was found to be sensitive, accurate, precise, simple, and rapid. Hence the present RP-HPLC method may be used for routine analysis of the raw materials, in combinational dosage formulations containing Amlodipine besylate and irbesartan. 14

15 ACKNOWLEDGEMENTS The authors are thankful to the Principal Dr. T.Y.Pasha, Parul institute of pharmacy & research,vadodara, for providing necessary facilities and Osaka Pharmaceutical ltd, Vadodara and Cadila pharmaceutical limited, Ankleshwar for providing the gift sample of Amlodipine besylate and irbesartan respectively. REFERENCES 1. United State Pharmacopoeia-2009, The Official Compendia of Std, Vol-II, pp Shethi P.D., HPLC-Quantitative analysis of pharmaceutical formulations; 1st Edn; CBS publishers & Distributors, New Delhi, 2001, pp International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use. Validation of Analytical : Text and Methodology ICH Q2 (R1) Patel Deval and Mehta Falgun A, Simultaneous Estimation of Amlodipine Besylate and Indapamide in Pharmaceutical Formulation by High Performance Liquid Chromatographic (RP-HPLC) Method. Sci. Pharm, March 2012, 80: Patil Priyanka R, Rakesh Sachin U, Dhabale P.N. and Burade K.B, RP- HPLC Method for Simultaneous Estimation of Losartan potassium and Amlodipine besylate in tablet Formulation. International Journal of ChemTech Research, July-Sept 2009,Vol.1, No.3, Patil Pournima S, More Harinath N. and Pishwikar Sachin A, RP-HPLC method for simultaneous estimation of Amlodipine besylate and Olmesartan medoxomil from tablet. International Journal of Pharmacy and Pharmaceutical Sciences, March 2011,vol-3,suppl-3, Prajapati Jignesh and Patel Ajay, Analytical method development and validation of Amlodipine besylate and Perindopril erbumine in combine dosage form by RP- HPLC. International Journal of PharmTech Research, April-June 2011 Vol. 3, No.2, Mhaske RA, Sahasrabudhe S and Mhaske AA, RP-HPLC method for simultataneous determination of irbesartan, losartan, hydro-chlorothiazide and chlorthalidone application to commercially available drug products. IJPSR,Aug 2012, Vol. 3, Issue Raju R. Ramesh and Bujji Babu N, Development and validation of HPLC method for the estimation of irbesartan in pharmaceutical dosage form. Pharmacophore, Feb 2011, Vol. 2 (2),